Process for the glycosylation of avermectin compounds

ABSTRACT

Avermectin compounds are glycosylated the 4&#39; and 4&#34;-positions by adding the avermectin compounds to the fermentation medium of Saccharapolyspora erythrea. The outer oleandrose sugar group of the avermectin compound is glycosylated with a glycosyl moiety, specifically a glucose group. In addition, other changes are effected in the avermectin moiety such as selective hydroxylation, epimerization at the 2-carbon and migration of the Δ 3-double bond to a Δ 2-position.

BACKGROUND OF THE INVENTION

Avermectin compounds are natural products produced by the fermentationof Streptomyces avermitilis as disclosed in U.S. Pat. No. 4,310,519 toAlbers-Schonberg et al. The avermectin compounds have a naturalα-L-oleandrosyl-α-L-oleandrosyloxy group at the 13-position. In U.S.Pat. No. 4,203,976 to Fischer et al certain synthetic procedures aredisclosed for glycosylting various hydroxy groups or the avermectinmolecule, including the 4"-hydroxy of the avermectin disaccharide group.The culture Saccharopolyspora erythrea identified in the culturecollection of Merck & Co., Inc. as MA 1625 is a known culture, publiclyavailable from the American Type Culture Collection at 12301 ParklawnDrive, Rockville, MD 20852, under the accession number ATCC 11635, andfurther described in Corcoran, Methods in Enzymology 43 pg 487-498(1975).

SUMMARY OF THE INVENTION

This invention is concerned with the preparation of avermectin compoundswith a glucose group substituted at the 4"-position of the natural,13-(α-L-oleandrosyl-α-L-oleandrosyloxy) group or at the 4'-position ofthe 13. (α-L-oleandrosyl) group of the avermectin monosaccharide whichare prepared by fermenting an avermectin compound in a culture medium ofSaccharopolyspora erythrea MA 1625, ATCC 11635. Thus, it is an object ofthis invention to describe the avermectin compounds prepared in suchfermentation medium. It is a further object of this invention todescribe the processes used the prepare such compounds. It is a stillfurther object to describe the antiparasitic uses of such compounds.Another object of this invention is to describe the additionalmodification of the avermectin compounds which are observed followingsuch fermentation. Additional objects will become apparent from areading of the following description.

DESCRIPTION OF THE INVENTION

This invention is concerned with the preparation of avermectintrisaccharide and disaccharide compounds where a glucose group is placedat the 4' and 4"-positions of an avermectin compound. The process iscarried out by culturing the microorganism Saccharopolyspora erythrea ina culture medium and adding the avermectin starting material to thefermentation broth. The culture S. erythrea is a well-knownmicroorganism that is readily available from the American Type CultureCollection under the accession number ATCC 11635. The morphological andcultural characteristics of S. erythrea are described in Int. J. Syst.Bacteriol, 37 pg 19-22 (1987), Int. J. Syst. Bacteriol, 30 pg 380 (1980)and Arch. Microbiol, 31 pg 353 (1958).

The process of the instant invention is best realized in the followingreaction scheme: ##STR1##

In the above reaction scheme the broken line at the 22,23-positionindicates a single or a double bond at the 22,23-position;

n is 0 or 1;

R₁ is present only when the broken line represents a single bond at the22,23-position and is hydrogen or hydroxy;

R₂ is alkyl of 1 to 8 carbon atoms, alkenyl of 2 to 8 carbon atoms orcycloalkyl at 3 to 8 carbon atoms;

R₃ is hydroxy or methoxy; and the broken line at the 2,3,4-positionsindicate a double bond at either the 2,3-position or at the3,4-position.

The above compounds of Formula II are novel compounds and are activeanthelmentic agents. They are to be considered as part of the instantinvention.

The instant process is carried out by adding a compound of Formula I tothe fermentation broth of S. erythrea and carrying out the fermentationas described below. The compound of Formula I can be added to thefermentation broth at any time during the fermentation period however ithas been found advantageous to add the starting material after allowingthe fermentation to proceed for a portion of its term but, to allow themicroorganism sufficient time to operate on the starting material,before the fermentation term is complete. Generally, the startingmaterial is added after the fermentation term is at least 10% completebut before it is 75% complete. Preferably the starting material is addedwhen the fermentation has completed from 20% to 50% of its scheduledterm.

The starting material is added to the fermentation broth in quantitiesof from 0.1 to 10 mg per liter of fermentation broth. Preferably thestarting material is added in quantities of from 1 to 8 mg per ml. offermentation broth.

The preferred compounds of the instant invention are realized when inthe above structural Formula II:

the broken line at the 22,23-position indicates a 22,23-single bond andR₁ is hydrogen;

R₂ is isopropyl or sec-butyl;

R₃ is hydroxy; and

the broken line at the 2,3,4-position indicates a 3,4-double bond.

The above described strain of Streptomyces erythrea MA-1625, ATCC 11635is illustrative of a strain which can be employed in the production ofthe instant compounds. However, the present invention also embracesmutants of the above described microorganism. For example, those mutantswhich are obtained by natural selection of those produced by mutatingagents including ionizing radiation such as ultraviolet irradiation, orchemical mutagens such as nitrosoguanidine or the like treatments arealso included within the ambit of this invention.

The instant compounds are produced during the aerobic fermentation ofsuitable aqueous nutrient media under conditions described hereinafter,with a producing strain of Streptomyces erythrea MA-1625, ATCC 11635.Aqueous media such as those used for the production of many antibioticsubstances are suitable for use in this process for the production ofthis macrocyclic compound. Such nutrient media contain sources of carbonand nitrogen assimilable by the microorganism and generally low levelsof inorganic salts. In addition, the fermentation media may containsmall amounts of inorganic salts and traces of metals necessary for thegrowth of the microorganisms, and production of the desired compounds.These are usually present in sufficient concentrations in the complexsources of carbon and nitrogen, which may be used as nutrient sources,but can, of course, be added separately to the medium if desired.

In general, carbohydrates such as sugars, for example dextrose, sucrose,maltose, lactose, dextran, cerelose, corn meal, oat flour, and the like,and starches are suitable sources of assimilable carbon in the nutrientmedia. The exact quantity of the carbon source which is utilized in themedium will depend, in part, upon the other ingredients in the medium,but it is usually found that an amount of carbohydrate between 0.5 and5% by weight of the medium is satisfactory. These carbon sources can beused individually or several such carbon sources may be combined in thesame medium.

Various nitrogen sources such as yeast hydrolysates, yeast autolysates,yeast cells, tomato paste, corn meal, oat flour, soybean meal, caseinhydrolysates, yeast extracts, corn steep liquors, distillers solubles,cottonseed meal, meat extract and the like, are readily assimilable bySaccharppolyspora erythrea MA-1625, ATCC 11635 in the production of theinstant compounds. The various sources of nitrogen can be used alone orin combination in amounts ranging from 0.2 to 6% by weight of themedium.

Among the nutrient inorganic salts, which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride,carbonate, and like ions. Also included are trace metals such as cobalt,manganese, and the like.

It should be noted that the media described hereinbelow and in theExamples are merely illustrative of the wide variety of media, which maybe employed, and are not intended to be limitative.

The following are Examples of media suitable for growing strains ofSaccharopolyspora erythrea MA-1625, ATCC 11635.

    ______________________________________                                        MEDIUM 1                                                                      Glucose                  5      g                                             Commerical Brown Sugar   10     g                                             Tryptone                 5      g                                             Yeast Extract            2.5    g                                             EDTA                     36     mg                                            (ethylene diamine                                                             tetracetic acid)                                                              betaine                  1.29   g                                             sodium propionate        0.11   g                                             distilled H.sub.2 O      1100   ml                                            pH 7.0-pH 7.2                                                                 MEDIUM 2                                                                      Sucrose                  15     g                                             Peptone                  5.0    g                                             Yeast extract            2.5    g                                             L-arginine               0.5    g                                             Distilled H.sub.2 O      1000   ml                                            pH 7.0                                                                        MEDIUM 3                                                                      Glucose                  50     g                                             NaCl                     5.0    g                                             (NH.sub.4).sub.2 SO.sub.4                                                                              2.0    g                                             CaCO.sub.3               6.0    g                                             propanol                 5      g                                             soya flour               30     g                                             distilled H.sub.2 O      1000   ml                                            MEDIUM 4                                                                      Soluble starch           15     g                                             Soytone                  20     g                                             CaCl.sub.2               0.1    g                                             yeast extract            1.5    g                                             soya oil                 50     ml                                            MOPS                     10 5   ml                                            Morpholino propane sulfonic acid)                                             MEDIUM 5                                                                      K.sub.2 HPO.sub.4        450    mg                                            saccharose               2.0    g                                             casein                   1.5    g                                             NaCl                     50     mg                                            L-arginine               15     mg                                            trace element mix A      1.0    ml                                            distilled water          1000   ml                                            pH 6.9                                                                        TRACE ELEMENT MIX                                                             Citric Acid              46.2   mg                                            FeSO.sub.4.7H.sub.2 O    2.0    mg                                            ZnSO.sub.4.7H.sub.2 O    1.0    mg                                            MnCl.sub.2.4H.sub.2 O    0.8    mg                                            CoCl.sub.2.6H.sub.2 O    0.1    mg                                            MgSO.sub.4.7H.sub.2 O    50     ml                                            Ascobic acid             0.12   mg                                            H.sub.2 O                160    ml                                            MEDIUM 6                                                                      Cottonseed oil           5.0    g                                             yeast extract            0.5    g                                             dextrose                 4.5    g                                             soybean oil              0.5    ul                                            CaCO3                    0.6    g                                             Trace element mix        1.0    ml                                            distilled H.sub.2 O      1000   ml                                            ______________________________________                                    

The fermentations employing Saccharopolyspora erythrea MA-1625, ATCC11635 can be conducted at temperatures ranging from about 20° C. toabout 40° C. For optimum results, it is most convenient to conduct thesefermentations at a temperature in the range of from about 24° C. toabout 30° C. Temperatures of about 27°-28° C. are most preferred. The pHof the nutrient medium suitable for producing the instant compounds canvary from about 5.0 to 8.5 with a preferred range of from about 6.0 to7.5.

Small scale fermentations are conveniently carried out by placingsuitable quantities of nutrient medium in a flask employing knownsterile techniques, inoculating the flask with either spores orvegetative cellular growth of Saccharopolyspora erythrea MA-1625, ATCC11635, loosely stoppering the flask with cotton and permitting thefermentation to proceed in a constant room temperature of about 30° C.on a rotary shaker at from 95 to 300 rpm for about 2 to 10 days. Forlarger scale work, it is preferable to conduct the fermentation insuitable tanks provided with an agitator and a means of aerating thefermentation medium. The nutrient medium is made up in the tank andafter sterilization is inoculated with a source of vegetative cellulargrowth of Saccharopolyspora erythrea MA-1625, ATCC 11635. Thefermentation is allowed to continue for from 1 to 8 days while agitatingand/or aerating the nutrient medium at a temperature in the range offrom about 24° to 37° C. The degree of aeration is dependent uponseveral factors such as the size of the fermentor, agitation speed, andthe like. Generally the larger scale fermentations are agitated at about95 to 500 RPM and about 50 to 500 liters per minute of air.

The novel compounds of this invention are found primarily in the aqueousportion of the fermentation medium on termination of the Streptomyceserythrea MA-1625, ATCC 11635 fermentation and may be removed andseparated therefrom as described below.

The separation of the novel compounds from the whole fermentation brothand the recovery of said compounds is carried out by solvent extractionand application of chromatographic fractionations with variouschromatographic techniques and solvent systems.

The instant compounds have slight solubility in water, but are solublein organic solvents. This property may be conveniently employed torecover the compound from the fermentation broth. Thus, in one recoverymethod, the whole fermentaion broth is combined with approximately anequal volume of an organic solvent. While any organic solvent may beemployed, it is preferable to use a water immiscible solvent such asethyl acetate, methylene chloride, chloroform, methyl ethyl ketone andthe like. Generally several extractions are desirable to achieve maximumrecovery. The solvent removes the instant compounds as well as othersubstances lacking the antiparasitic activity of the instant compounds.If the solvent is a water immiscible one, the layers are separated andthe organic solvent is evaporated under reduced pressure. If the solventis water miscible, it can be extracted with a water immiscible solventto separate the entrained water. This solvent can then be concentratedunder reduced pressure. The residue is placed onto a chromatographycolumn containing preferably, silica gel. The column retains the desiredproducts and some impurities, but lets many of the impurities,particularly the nonpolar impurities, pass through. The column is washedwith a moderately polar organic solvent such as methylene chloride,chloroform or hexane to further remove impurities, and is then washedwith a mixture of methylene chloride, chloroform or hexane and anorganic solvent of which acetone, ethyl acetate, methanol, and ethanoland the like are preferred. The solvent is evaporated and the residuefurther chromatographed using column chromatography, thin layerchromatography, preparative layer chromatography, high pressure liquidchromatography and the like, with silica gel, aluminum oxide, dextrangels and the like, as the chromatographic medium, with various solventsand combinations of solvents as the eluent. Thin layer, high pressure,liquid and preparative layer chromatography may be employed to detectthe presence of, and to isolate the instant compounds. The use of theforegoing techniques as well as other known to those skilled in the art,will afford purified compositions containing the instant compounds. Thepresence of the desired compounds is determined by analyzing the variouschromatographic fractions for biological activity against selectedparasites, or physicochemical characteristics. The structures of theinstant compounds has been determined by detailed analysis of thevarious spectral characteristics of the compounds, in particular theirnuclear magnetic resonance, mass, ultraviolet and infrared spectra.

The instant compounds are potent endo-and ecto-antiparasitic agentsagainst parasites particularly helminths, ectoparasites, insects, andacarides, infecting man, animals and plants, thus having utility inhuman and animal health, agriculture and pest control in household andcommercial areas.

The disease or group of diseases described generally as helminthiasis isdue to infection of an animal host with parasitic worms known ashelminths. Helminthiasis is a prevalent and serious economic problem indomesticated animals such as swine, sheep, horses, cattle, goats, dogs,cats, fish, buffalo, camels, llamas, reindeer, laboratory animals,furbearing animals, zoo animals and exotic species and poultry. Amongthe helminths, the group of worms described as nematodes causeswidespread and often times serious infection in various species ofanimals. The most common genera of nematodes infecting the animalsreferred to above are Haemonchus, Trichostrongylus, Ostertagia,Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Habronema,Druschia, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma,Uncinaria, Toxascaris and Parascaris. Certain of these, such asNematodirus, Cooperia, and Oesophagostomum attack primarily theintestinal tract while others, such as Haemonchus and Ostertagia, aremore prevalent in the stomach while still others such as Dictyocaulusare found in the lungs. Still other parasites may be located in othertissues and organs of the body such as the heart and blood vessels,subcutaneous and lymphatic tissue and the like. The parasitic infectionsknown as helminthiases lead to anemia, malnutrition, weakness, weightloss, severe damage to the walls of the intestinal tract and othertissues and organs and, if left untreated, may result in death of theinfected host. The compounds of this invention have unexpectedly highactivity against these parasites, and in addition are also activeagainst Dirofilaria in dogs and cats, Nematospiroides, Syphacia,Aspiculuris in rodents, arthropod ectoparasites of animals and birdssuch as ticks, mites, lice, fleas, blowflies, in sheep Lucilia sp.,biting insects and such migrating diperous larvae as Hypoderma sp.cattle, Gastrophilus in horses, and Cuterebra sp. in rodents andnuisance flies including blood feeding flies and filth flies.

The instant compounds are also useful against parasites which infecthumans. The most common genera of parasites of the gastro-intestinaltract of man are Ancylostoma, Necator, Ascaris, Strongyloides,Trichinella, Capillaria, Trichuris, and Enterobius. Other medicallyimportant genera of parasites which are found in the blood or othertissues and organs outside the gastrointestinal tract are the filiarialworms such as Wuchereria, Brugia, Onchocerca and Loa, Dracunuculus andextra intestinal stages of the intestinal worms Strongyloides andTrichinella. The compounds are also of value against arthropodsparasitizing man, biting insects and other dipterous pests causingannoyance to man.

The compounds are also actove against household pests such as thecockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle,Attagenus sp., the housefly Musca domestica as well as fleas, house dustmites, termites and ants.

The compounds are also useful against insect pests of stored grains suchas Tribolium sp., Tenebrio sp. and of agricultural plants such asaphids, (Acyrthiosiphon sp.); against migratory orthopterans such aslocusts and immature stages of insects living on plant tissue. Thecompounds are useful as a nematocide for the control of soil nematodesand plant parasites such as Meloidogyne sp. which may be of importancein agriculture. The compounds are also highly useful in treating acerageinfested with fire ant nests. The compounds are scattered above theinfested area in low levels in bait formulations which are broght backto the nest. In addition to a direct-but-slow onset toxic effect on thefire ants, the compound has a long-term effect on the nest bysterilizing the queen which effectively destroys the nest.

The compounds of this invention may be administered in formulationswherein the active compound is intimately admixed with one or more inertingredients and optionally indlucing one or more additiona activeingredients. The compounds may be used in any composition known to thoseskilled in the art for administration to humans and animals, forapplication to plants and for premise and area application to controlhousehold pests in either a residential or commercial setting. Forapplication to humans and animals to control internal and externalparasites, oral formulations, in solid or liquid or parenteral liquid,implant or depot injection forms may be used. For topical applicationdip, spray, powder, dust, pour-on, spot-on, jetting fluid, shampoos,collar, tag or harness, may be used. For agricultural premise or areaapplications, liquid spray, powders, dust, or bait forms may be used. Inaddition "feed-through" forms may be used to control nuisance flies thatfeed or breed in animal waste. The compounds are formulated, such as byencapsulation, to lease a residue of active agent in the animal wastewhich controls filth flies or other arthropod pests.

These compounds may be administered orally in a unit dosage form such asa capsule, bolus or tablet, or as a liquid drench where used as ananthelmintic in mammals. The drench is normally a solution, suspensionor dispersion of the active ingredient usually in water together with asuspending agent such as bentonite and a wetting agent or likeexcipient. Generally, the drenches also contain an antifoaming agent.Drench formulations generally contain from about 0.001 to 0.5% by weightof the active compound. Preferred drench formulations may contain from0.01 to 0.1% by weight. The capsules and boluses comprise the activeingredient admixed with a carrier vehicle such as starch, talc,magnesium stearate, or di-calcium phosphate.

Where it is desired to administer the instant compounds in a dry, solidunit dosage form, capsules, boluses or tablets containing the desiredamount of active compound usually are employed. These dosage forms areprepared by intimately and uniformly mixing the active ingredient withsuitable finely divided diluents, fillers, disintegrating agents, and/orbinders such as starch, lactose, talc, magnesium stearate, vegetablegums and the like. Such unit dosage formulations may be varied widelywith respect to their total weight and content of the antiparasiticagent depending upon factors such as the type of host animal to betreated, the severity and type of infection and the weight of the host.

When the active compound is to be administered via an animal feedstuff,it is intimately dispersed in the feed or used as a top dressing or inthe form of pellets or liquid which may then be added to the finishedfeed or optionally fed separately. Alternatively, feed based individualdosage forms may be used such as a chewable treat. Alternatively, theantiparasitic compounds of this invention may be administered to animalsparenterally, for example, by intraruminal, intramuscular,intravascular, intratracheal, or subcutaneous injection in which theactive ingredient is dissolved or dispersed in a liquid carrier vehicle.For parenteral administration, the active material is suitably admixedwith an acceptable vehicle, preferably of the vegetable oil variety suchas peanut oil, cotton seed oil and the like. Other parenteral vehiclessuch as organic preparation using solketal, glycerol formal, propyleneglycol, and aqueous parenteral formulations are also used. The activecompound or compounds are dissolved or suspended in the parenteralformulation for administration; such formulations generally contain from0.0005 to 5% by weight of the active compound.

Although the antiparasitic agents of this invention find their primaryuse in the treatment and/or prevention of helminthiasis, they are alsouseful in the prevention and treatment of diseases caused by otherparasites, for example, arthropod parasites such as ticks, lice, fleas,mites and other biting arthropods in domesticated animals and poultry.They are also effective in treatment of parasitic diseases that occur inother animals including humans. The optimum amount to be employed forbest results will, of course, depend upon the particular compoundemployed, the species of animal to be treated and the type and severityof parasitic infection or infestation. Generally good results areobtained with our novel compounds by the oral administration of fromabout 0.001 to 10 mg per kg of animal body weight, such total dose beinggiven at one time or in divided doses over a relatively short period oftime such as 1-5 days. With the preferred compounds of the invention,excellent control of such parasites is obtained in animals byadministering from about 0.025 to 0.5 mg per kg of body weight in asingle dose. Repeat treatments are given as required to combatre-infections and are dependent upon the species of parasite and thehusbandry techniques being employed. The techniques for administeringthese materials to animals are known to those skilled in the veterinaryfield.

When the compounds described herein are administered as a component ofthe feed of the animals, or dissolved or suspended in the drinkingwater, compositions are provided in which the active compound orcompounds are intimately dispersed in an inert carrier or diluent. Byinert carrier is meant one that will not react with the antiparasiticagent and one that may be administratered safely to animals. Preferably,a carrier for feed administration is one that is, or may be, aningredient of the animal ration.

Suitable compositions include feed premixes or supplements in which theactive ingredient is present in relatively large amounts and which aresuitable for direct feeding to the animal or for addition to the feedeither directly or after an intermediate dilution or blending step.Typical carriers or diluents suitable for such compositions include, forexample, distillers' dried grains, corn meal, citrus meal, fermentationresidues, ground oyster shells, wheat shorts, molasses solubles, corncob meal, edible bean mill feed, soya grits, crushed limestone and thelike. The active compounds are intimately dispersed throughout thecarrier by methods such as grinding, stirring, milling or tumbling.Compositions containing from about 0.005 to 2.0% weight of the activecompound are particularly suitable as feed premixes. Feed supplements,which are fed directly to the animal, contain from about 0.0002 to 0.3%by weight of the active compounds.

Such supplements are added to the animal feed in an amount to give thefinished feed the concentration of active compound desired for thetreatment and control of parasitic diseases. Although the desiredconcentration of active compound will vary depending upon the factorspreviously mentioned as well as upon the particular compound employed,the compounds of this invention are usually fed at concentrations ofbetween 0.00001 to 0.002% in the feed in order to achieve the desiredanti-parasitic result.

In using the compounds of this invention, the individual compounds maybe prepared and used in that form. Alternatively, mixtures of theindividual compounds may be used, or other active compounds not relatedto the compounds of this invention.

The compounds of this invention are also useful in combattingagricultural pests that inflict damage upon crops while they are growingor while in storage. The compounds are applied using known techniques assprays, dusts, emulsions and the like, to the growing or stored crops toeffect protection from such agricultural pests.

The following examples are provided in order that this invention mightbe more fully understood; they are not to be construed as limitative ofthe invention.

EXAMPLE 1 1-4"-O-Glucosyl ivermectin

S. erythraea (ATCC 11635) was grown in medium M102 as described byCorcoran (Methods in Enzymology 43: 487-498 1975). It contained thefollowing in 1000 ml of distilled water: glucose, 5 g; commercial brownsugar (Domino's), 10 g; tryptone, 5 g; yeast extract, 2.5 g; ethylenediamine tetraacetate, 0.036 g; betaine, 1.2 g; sodium propionate, 0.11g. The medium was adjusted to pH 7.0-7.2 and 2 ml of trace elementssolution which contained the following in g/l were added

FeCl₃.6H₂ O, 0.2; ZnCl₂, 0.04; MnCl₂.4H₂ O; 0.01; CuCl₂.2H₂ O, 0.01;NaB₄ O₇.10H₂ O, 0.01; (NH₄)₆ Mo₇ O₂₄.4H₂ O, 0.01.

INOCULUM PREPARATION

Frozen vegetative mycelia (FVM) were prepared by inoculating 250 mlmedium 102 in a 2 liter 3 baffle flask and incubating at 32° C., 85%relative humidity and 200 RPM for 48 hours. The packed cell volume ofthe culture was 10% and the pH 6.9. Aliquots of the culture was frozenand used as source of inoculum for future experiments.

SEED CULTURE

To 40 ml of medium M102 in a 250 ml flask, 1.0 ml of FVM was added asinoculum and the flasks were incubated at 30° C., 85% relative humidityand 200 RPM for 40 hours.

BIOTRANSFORMATION AND ISOLATION

To 40 ml of medium M102 in a 250 ml flask, 1.0 ml of seed culture wasadded and the flasks were incubated at 30° C., 85% relative humidity at200 RPM for 24 hours. 2.5 g of ivermectin (22,23 dihydro avermectinBla/Blb) in 0.1 ml DMSO were added and the flasks were incubated asabove for 5 days. Each flasks was extracted with 2×80 ml portions of CH₂Cl₂. The CH₂ Cl₂ extracts were combined, concentrated and theavermectins were partially purified by preparative TLC on silica gel 50using methylene chloride: ethylacetate: methanol (9.9:1) as the solvent.The individual avermectin bands were eluted from the silica,concentrated and further purified by HPLC on Dupont Zorbax ODS using CH₃OH:H₂ O 90:10, 85:15 80:20 or 70:30) as the mobile phase. The structuresof the purified avermectins were determined by mass spectroscopy and NMRspectroscopy

The HPLC retention times of 4"-O-glucosyl ivermectin on a Dupont ZorbaxODS column with CH₃ OH:H₂ O at 1 ml/min as the mobile phase is 7.2 minat 90:10, 18.7 min at 85:15 at 40.9 min at 80:20. The molecular weightdetermined by mass spectroscopy is 1036. Characteristic NMR spectroscopysignals of this derivative are: 4.45d, J=7, H-1 (glucose), ca 3.37 m.H-2 (glucose). This compound is nearly as potent an anthelmintic asinvermectin, but is greater than 10-fold safer.

EXAMPLE 2 4"-O-Glucosyl avermectin Bla

Procedure was the same as example 1 except 2.5 mg of avermectin Bla in0.1 ml DMSO were added to the biotransformation flasks. The HPLCretention times on a Dupont Zorbax ODS column with CH₃ OH:H₂ O at 1mg/min as the mobile phase are 5.5 min at 90:10, 10.2 min at 85:15 and20:3 min at 80:20. The molecular weight determined by mass spectroscopyis 1034. Characteristic NMR spectroscopy signal are: 4.45H,8 H1(glucose), 3.38 m. H-2 (glucose).

This derivative is nearly as potent an anhelmintic and insecticide asavermectin Bla but is greater than 10 fold safer.

EXAMPLE 3 4"-O-Glucosyl 22,23 dihydro avermectin Blb

Procedure was the same as example 1 except 0.5 mg of 22,23 dihydroavermectin Blb was added to the biotransformation flasks. The HPLCretention times on a Dupont Zorbax ODS column with CH₃ OH:H₂ O at 1ml/min as the mobile phase are 6.4 min at 90:10, 15.2 min at 85:15 at32.5 min at 80:20. The molecular weight determined by mass spectroscopyof 1024.

EXAMPLE 4 4"-O-Glucosyl avermectin Blb

Procedure was the same as example 1 except 0.5 mg of avermectin Blb wasadded to the biotransformation flasks. The HPLC retention times on aDupont Zorbax ODS column with CH₃ OH:H₂ O at 1 mg/min as the mobilephase are 5.0 min at 90:10, 8.75 min at 85:15, 15.87 min at 80:20. Themolecular weight determined by mass spectroscopy of 1022. CharacteristicNMR signals are: 4.43d, 7.5. H 1. glucose; methyl doublets at 0.92 (6H).1.09, 1.74, 1.22 and 1.31 (last two represent olendrose methyls).

EXAMPLE 5 4'-O-Glucosyl 22,23 dihydro avermectin Bla/Blb monosaccharide

Procedure was the same as example 1 except 2.5 mg of 22,23-dihydroavermectin Bla/Blb monosaccharide were added to the biotransformationflasks. The HPLC retention times of this derivative on a Dupont ZorbaxODS column with CH₃ OH:H₂ O at 1 ml/min the mobile phase are 11.3 min at85:15, and 20.7 min at 80:20. The molecular weight determined by massspectroscopy of 892. Characteristic NMR spectroscopy peaks are: 4.62d.J=8, H-1(glucose), (glucose). 3.18dd. J=10,8. H-2 (glucose).

EXAMPLE 6 4'-O-Glucosyl avermectin Bla monosaccharide

Procedure was the same as example 1 except 2.5 mg of avermectin Blamonosaccharide was added to the biotransformation flasks. The HPLCretention times of this derivative on a Dupont Zorbax ODS column withCH₃ OH:H₂ O as mobile phase are 6.61 min at 85:15, at 80:20. Themolecular weight determined by mass spectroscopy of 890. CharacteristicNMR spectroscopy peaks are: 4.62d. J=8, H-1 (glucose), 3.18dd, 10.8. H-2(glucose).

What is claimed is:
 1. A process for the preparation of a compoundhaving the formula: ##STR2## wherein the broken line at the 22,23-position indicates a single or a double bond at the 22, 23-position;nis 1 or 2 R₁ is present only when the broken line represents a singlebond at the 22, 23-position and is hydrogen or hydroxy; R₂ is alkyl of 1to 8 carbon atoms, alkenyl of 2 to 8 carbon atoms or cycloalkyl of 3 to8 carbon atoms; R₃ is hydroxy or methoxy; and the broken line at the2,3,4-position indicates a double bond at either the 2,3-position or the3,4-position, which comprises fermenting in a nutrient medium containingsources of carbon, sources of nitrogen and inorganic salts,Saccharopolysporaerythrea ATCC 11635, at a pH of from 5 to 8.5, atemperature of from 20° to 40° C., for from 2 to 10 days, from 0.1 to 10mg/ml of fermentation medium a compound having the formula: ##STR3##where n, R₁, R₂, and R₃ are as defined above and recovery of theproducts from the fermentation medium.
 2. The process of claim 1 wherethe broken line at the 22,23-position indicates a 22,23-single bond andR₁ is hydrogen;n is 2; R₂ is isopropyl or sec-butyl; R₃ is hydroxy; andthe broken line at the 2,3,4-position indicates a 3,4-double bond. 3.The process of claim 1 where the starting material is added to thefermentation medium when the fermentation has completed from 10 to 75%of the scheduled term of 2 to 10 days.
 4. The process of claim 3 wherethe starting material is added to the fermentation medium when thefermentation has completed from 20 to 50% of the scheduled term of 2 to10 days.
 5. The process of claim 1 where the starting material is addedto the fermentation medium in a quantity of from 1 to 8 mg per ml. offermentation medium.